Fluid delivery system and method

ABSTRACT

A method and apparatus for delivering one or more fluids. Fluids may be delivered sequentially from a common vessel to a chemical, biological or biochemical process.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.10/587,156, filed May 16, 2007, which is a U.S. National Application ofInternational Application No. PCT/US2005/003514, filed Jan. 26, 2005,which claims benefit under 35 U.S.C. §119(e) of U.S. ProvisionalApplication Ser. No. 60/539,358, filed Jan. 26, 2004, U.S. ProvisionalApplication Ser. No. 60/539,416, filed Jan. 26, 2004 and U.S.Provisional Application Ser. No. 60/565,866, filed Apr. 26, 2004, all ofwhich are which are incorporated herein by reference.

GOVERNMENT FUNDING

This invention was made with government support under GM051559 awardedby National Institutes of Health and under ECS-0004030 awarded byNational Science Foundation. The government has certain rights in theinvention.

BACKGROUND OF INVENTION

1. Field of Invention

This invention relates to a method and apparatus for the delivery and/orstorage of one or more fluids and, in particular, to a method andapparatus for storing and delivering chemical and biological reagents.

2. Discussion of Related Art

The delivery of fluids plays an important role in fields such aschemistry, microbiology and biochemistry. These fluids may includeliquids or gases and may provide reagents, solvents, reactants, orrinses to chemical or biological processes. Often, more than one fluidis delivered to a reaction vessel or site to promote interaction betweenthe fluids or components of the fluids. Intermittent rinse fluids mayalso be used to remove unwanted reactants or to prepare a reactor,reaction site or assay site.

While various microfluidic devices and methods, such as microfluidicassays, can provide inexpensive, sensitive and accurate analyticalplatforms, fluid delivery to the platform can add a level of cost andsophistication that may require testing to be performed in a laboratoryrather than in the field, where it may be most useful.

As chemical and biochemical platforms become smaller due to improvementsin areas such as microfluidics, smaller reagent quantities are requiredto do a similar number of assays or reactions. Typically, however,smaller size platforms do not diminish the need to supply multiplereagents and rinses to a reaction site. For instance, some microfluidicassays may require less than a microliter of reagent fluids, but two,three or more different fluids may need to be supplied in accuratequantities and in proper sequence.

For microfluidic assays and reactors, fluids are often supplied by anoperator using a micropipette. A fluid may be pipetted into an inlet ofa microfluidic system and the fluid may be drawn through the system byapplication of a vacuum source to the outlet end of the microfluidicsystem. Reagents may also be pumped in, for instance by using differentsyringe pumps filled with the required reagents. After one fluid ispumped into the microfluidic device, a second can be pumped in bydisconnecting a line from the first pump and connecting a line from asecond pump. Alternatively, valving may be used to switch from onepumped fluid to another. Different pumps are used for each fluid toavoid cross contamination. This may be of particular relevance when twofluids contain components that may react with each other or, when mixed,can affect the results of an assay or reaction.

Continuous flow systems may use a series of two different fluids passingserially through a reaction channel. Fluids can be pumped into a channelin serial fashion by switching, through valving, the fluid source thatis feeding the tube. The fluids constantly move through the system insequence and are allowed to react in the channel. For example, a PCRreaction can be run using continuous flow. See Obeid et al.,“Microfabrication Device for DNA and RNA Amplification byContinuous-Flow Polymerase Chain Reaction and ReverseTranscription-Polymerase Chain Reaction with Cycle Number Selection,”Analytical Chemistry, 2003, 75, 288-295.

The utility of fluid systems may be affected by the storage time, orshelf life, of any reagents that are to be used with a system. Aportable microfluidic system can be transported to almost any location,but when reagents must be freshly prepared, the usefulness of the systemin the field can be diminished. This may be true in particular forbiological and biochemical based systems that may rely on reagents that,for example, are unstable, have short shelf lives or must be storedunder special conditions, such as refrigeration.

An accurate early and ongoing determination of a disease condition isimportant for the prevention and treatment of human and animal diseases.One class of diagnostic techniques uses immunoassay reactions to detectthe presence of either an antigen or an antibody in a sample taken froma subject. These immunoassay methods include, for example, ELISA,immunochromatographic assays (strip tests, dipstick assays and lateralflow assays), and sandwich assays. Accuracy, reliability, and ease ofuse of these types of assays has improved, but often testing requireslaboratory conditions, power supplies, and training that may not beavailable in some areas where testing is desired.

One type of sandwich assay uses gold conjugated antibodies to enhancedetection. For example, see PCT publication WO/91/01003. Enhancement ofa gold colloid signal can be achieved by staining the gold colloids withsilver. First, an antigen is immobilized onto a solid polystyrenesubstrate. A human anti-HIV antibody is then captured by the antigen andis therefore itself immobilized on the substrate. The antibody is thenexposed to anti-human IgG labeled with a colloidal gold particle andthus labeled IgG becomes bonded to the antibody. Theantigen-antibody-IgG complex is then exposed to a solution containingsilver ions and these become nucleated around the gold particles assolid silver particles having a dark color to the eye.

The development of microfluidics and microfluidic techniques hasprovided improved chemical and biological research tools, includingplatforms for performing chemical reactions, combining and separatingfluids, diluting samples, and generating gradients. For example, seeU.S. Pat. No. 6,645,432, hereby incorporated by reference herein.

SUMMARY OF INVENTION

In one aspect, the invention is a method, the method comprisingproviding a first and a second fluid maintained separately from eachother in a common vessel, transferring the first and second fluids inseries from the vessel to a reaction site to carry out a predeterminedchemical or biochemical reaction, and avoiding contact between the firstand second fluids, at least until after the fluids have been applied tothe reaction site.

In another aspect, an apparatus is provided, the apparatus comprising asealed vessel, a first static fluid disposed in the vessel, a secondstatic fluid disposed in the vessel, and a third static fluid disposedin the vessel, wherein the third fluid separates the first and secondfluids, and at least the first and second fluids are selected for use ina predetermined chemical or biological reaction in a predeterminedsequence.

In another aspect, another method is provided, the method comprisingflowing a first fluid into a vessel, flowing a second fluid into thevessel, the second fluid being substantially immiscible with the firstfluid, flowing a third fluid into the vessel, wherein the third fluid issubstantially immiscible with the second fluid and wherein the thirdfluid is not contacting the first fluid, and sealing the fluids in thevessel.

In another aspect another apparatus is provided, the apparatuscomprising a sealed vessel comprising a chamber, defining a continuousvoid, containing a first fluid and a second fluid, the first and secondfluids constructed and arranged to be deliverable from the vesselseparately for sequential use in a predetermined chemical or biologicalreaction wherein the sealed vessel is constructed and arranged forstoring the first and second fluids for at least one hour prior to useof the first and second fluids in the predetermined chemical orbiological reaction.

In another aspect an assay kit is provided, the kit comprising a surfaceincluding a microfluidic channel, at least one of an antibody or anantigen associated with a portion of the microfluidic channel, a vessel,a first static fluid disposed in the vessel, the first static fluidcomprising a metal colloid associated with an antibody or an antigen, asecond static fluid disposed in the vessel, the second static fluidcomprising a metal precursor, a third static fluid disposed in thevessel wherein the third fluid separates the first and second fluids,and instructions for performing the assay.

In another aspect, another method is provided, the method comprisingproviding a first and a second fluid statically maintained separatelyfrom each other in a common vessel for greater than one minute, applyingin series the first and second fluid to a reaction site, and avoidingcontact between the first and second fluids, at least until after thefluids have been applied to the reaction site.

In another aspect a method is provided, the method comprising providinga first and a second fluid maintained separately from each other in acommon vessel, transferring the first and second fluids in series fromthe vessel to a reaction site to carry out a predetermined chemical orbiochemical reaction, allowing a component of the first fluid to becomeassociated with the reaction site, and allowing a component of thesecond fluid to become associated with the component of the first fluid.

BRIEF DESCRIPTION OF DRAWINGS

The accompanying drawings, are not intended to be drawn to scale. In thedrawings, each identical or nearly identical component that isillustrated in various figures is represented by a like numeral. Forpurposes of clarity, not every component may be labeled in everydrawing. In the drawings:

FIG. 1 is an illustration of one embodiment of an assay of theinvention;

FIG. 2 is an illustration of an assay including a detector;

FIG. 3 is a schematic illustration of an optical detector;

FIG. 4 is a graph illustrating absorbance versus analyte concentration;

FIG. 5 illustrates graphically and in a photocopy of a micrograph theamount of opaque material present at high and low analyteconcentrations;

FIG. 6 provides photocopies of micrographs showing the formation ofopaque material at various analyte concentrations;

FIG. 7 provides graphical data regarding four different assaytechniques;

FIG. 8 provides graphical data indicating absorbance vs. time ofexposure and provides photocopies of micrographs showing an opaquematerial;

FIG. 9 provides a side view of an assay detection system;

FIG. 10 provides graphical data comparing apparent absorbance by twodifferent techniques;

FIG. 11 provides additional graphical data comparing absorbance by twodifferent techniques;

FIG. 12 is a schematic illustration of a vessel containing fluid plugs;

FIG. 13 illustrates a technique for filling a vessel;

FIG. 14 illustrates another technique for filling a vessel;

FIG. 15 illustrates one embodiment of delivering a series of fluids toan assay device;

FIGS. 16 a, b and c illustrate graphically the fluorescence response toa series of sequentially applied fluid plugs;

FIG. 17 provides a schematic illustration of various fluid reagent plugsin a vessel;

FIG. 18 illustrates a method for making a microfluidic assay device.

FIG. 19 provides a schematic illustration of associated components fromthe reagent fluids of the vessel of FIG. 17;

FIG. 20 is a photocopy of a fluorescent micrograph of an assay completedon the device of FIG. 18;

FIG. 21 provides another illustration of various fluid reagent plugs ina vessel;

FIG. 22 illustrates an embodiment showing a change in response thatvaries with a change in antibody concentration; and

FIG. 23 illustrates graphically a change in fluorescence againstcartridge storage time.

DETAILED DESCRIPTION

This invention is not limited in its application to the details ofconstruction and the arrangement of components set forth in thefollowing description or illustrated in the drawings. The invention iscapable of other embodiments and of being practiced or of being carriedout in various ways. Also, the phraseology and terminology used hereinis for the purpose of description and should not be regarded aslimiting. The use of “including,” “comprising,” or “having,”“containing”, “involving”, and variations thereof herein, is meant toencompass the items listed thereafter and equivalents thereof as well asadditional items.

The invention relates to a method and apparatus for the delivery offluids. The term “fluid” is used herein, as it is commonly by thoseskilled in the art, to include both liquids and gases, including gaseousmixtures. Also included are aqueous and non-aqueous solvents, solutionsand suspensions.

A “reaction site” is a location where a chemical, physical orbiochemical process occurs. These processes may include any of, forexample, chemical reactions, electrochemical photochemical reactions,chemical and biological assays such as disease condition assessment,immunoassays, nucleic acid binding and/or identification, and proteinbinding and/or identification. Also included are finishing processes,surface treatments and phase-altering reactions.

As used herein, “immiscible” is used according to its common meaning inthe art. Specifically, a first fluid is immiscible in a second fluid ifthe first fluid is not substantially soluble in the second fluid. Insome instances, a first fluid may be immiscible in a second fluid if itis less than 0.1%, less than 1%, less than 10% or less than 50% solublein a second fluid under environmental conditions at which the fluids arestored or used.

“Integral article” means a single piece of material, or assembly ofcomponents integrally connected with each other. As used herein, theterm “integrally connected,” when referring to two or more objects,means objects that do not become separated from each other during thecourse of normal use, e.g., cannot be separated manually; separationrequires at least the use of tools, and/or by causing damage to at leastone of the components, for example, by breaking, peeling, etc.(separating components fastened together via adhesives, tools, etc.).

“Instructions” can and often do define a component of promotion, andtypically involve written instructions on or associated with packagingof compositions of the invention, optionally as part of a kit.Instructions also can include any oral or electronic instructionsprovided in any manner. The “kit” typically, and preferably defines apackage including both any one or a combination of the components ordevices of the invention and the instructions, but can also includecomponents or devices of the invention and instructions of any form thatare provided in connection with the components or devices in a mannersuch that a clinical professional will clearly recognize that theinstructions are to be associated with the components or devices.

In some, but not all embodiments, all or some of the components of thesystems and methods described herein are microfluidic. “Microfluidic,”as used herein, refers to a device, apparatus or system including atleast one fluid channel having a cross-sectional dimension of less than1 mm, and a ratio of length to largest cross-sectional dimension of atleast 3:1. A “microfluidic channel,” as used herein, is a channelmeeting these criteria.

The “cross-sectional dimension” of the channel is measured perpendicularto the direction of fluid flow. Most fluid channels in components of theinvention have maximum cross-sectional dimensions less than 2 mm, and insome cases, less than 1 mm. In one set of embodiments, all fluidchannels containing embodiments of the invention are microfluidic orhave a largest cross sectional dimension of no more than 2 mm or 1 mm.In another embodiment, the fluid channels may be formed in part by asingle component (e.g. an etched substrate or molded unit). Of course,larger channels, tubes, chambers, reservoirs, etc. can be used to storefluids in bulk and to deliver fluids to components of the invention. Inone set of embodiments, the maximum cross-sectional dimension of thechannel(s) containing embodiments of the invention are less than 500microns, less than 200 microns, less than 100 microns, less than 50microns, or less than 25 microns.

A “channel,” as used herein, means a feature on or in an article(substrate) that at least partially directs the flow of a fluid. Thechannel can have any cross-sectional shape (circular, oval, triangular,irregular, square or rectangular, or the like) and can be covered oruncovered. In embodiments where it is completely covered, at least oneportion of the channel can have a cross-section that is completelyenclosed, or the entire channel may be completely enclosed along itsentire length with the exception of its inlet(s) and outlet(s). Achannel may also have an aspect ratio (length to average cross sectionaldimension) of at least 2:1, more typically at least 3:1, 5:1, or 10:1 ormore. An open channel generally will include characteristics thatfacilitate control over fluid transport, e.g., structuralcharacteristics (an elongated indentation) and/or physical or chemicalcharacteristics (hydrophobicity vs. hydrophilicity) or othercharacteristics that can exert a force (e.g., a containing force) on afluid. The fluid within the channel may partially or completely fill thechannel. In some cases where an open channel is used, the fluid may beheld within the channel, for example, using surface tension (i.e., aconcave or convex meniscus).

The channel may be of any size, for example, having a largest dimensionperpendicular to fluid flow of less than about 5 mm or 2 mm, or lessthan about 1 mm, or less than about 500 microns, less than about 200microns, less than about 100 microns, less than about 60 microns, lessthan about 50 microns, less than about 40 microns, less than about 30microns, less than about 25 microns, less than about 10 microns, lessthan about 3 microns, less than about 1 micron, less than about 300 nm,less than about 100 nm, less than about 30 nm, or less than about 10 nm.In some cases the dimensions of the channel may be chosen such thatfluid is able to freely flow through the article or substrate. Thedimensions of the channel may also be chosen, for example, to allow acertain volumetric or linear flowrate of fluid in the channel. Ofcourse, the number of channels and the shape of the channels can bevaried by any method known to those of ordinary skill in the art. Insome cases, more than one channel or capillary may be used. For example,two or more channels may be used, where they are positioned inside eachother, positioned adjacent to each other, positioned to intersect witheach other, etc.

A “plug” is defined herein as a continuous volume of a first fluid, theboundaries of which are defined by a wall or walls of a vessel and oneor more interfaces with a second fluid that is substantially immisciblewith the first fluid. An example of a plug would be a one microlitervolume of an aqueous solution in a capillary tube bounded by air at bothends of the one microliter volume. Another example of a plug would be aone milliliter volume of a non-aqueous liquid in a sealed length oftubing, the non-aqueous fluid being bounded at one end by the sealed endof the tubing and at the opposing end by an aqueous liquid.

If a fluid is “statically maintained” in a vessel, the fluid does notchange its position in relation to the vessel although it may, forexample, expand or contract or vibrate in its statically maintainedposition. The vessel containing the fluid may be moved or re-orientedwhile the fluid is statically maintained.

If a fluid is of the same “type” as a second fluid, it means that thetwo fluids serve the same purpose in an assay or reaction, although theymay be of different volumes. For example, two different rinse solutionswould be considered the same type of solution while a solution includinga reagent would not be of the same type as a rinse solution.

If two fluids are “distinct” from each other, they are not intermixedand fill separately distinguishable volumes. For instance, two fluidsmay be distinct if they are immiscible or if they are physicallyseparated, such as by a separation fluid.

The term “binding” refers to the interaction between a correspondingpair of molecules that exhibit mutual affinity or binding capacity,typically specific or non-specific binding or interaction, includingbiochemical, physiological, and/or pharmaceutical interactions.Biological binding defines a type of interaction that occurs betweenpairs of molecules including proteins, nucleic acids, glycoproteins,carbohydrates, hormones and the like. Specific examples includeantibody/antigen, antibody/hapten, enzyme/substrate, enzyme/inhibitor,enzyme/cofactor, binding protein/substrate, carrier protein/substrate,lectin/carbohydrate, receptor/hormone, receptor/effector, complementarystrands of nucleic acid, protein/nucleic acid repressor/inducer,ligand/cell surface receptor, virus/ligand, etc.

An “opaque material” is a substance that interferes with thetransmittance of light at one or more wavelengths. An opaque materialdoes not merely refract light, but reduces the amount of transmissionthrough the material by, for example, absorbing or reflecting light.Different opaque materials or different amounts of an opaque materialmay allow transmittance of less than 90, 80, 70, 60, 50, 40, 30, 20, 10or 1 percent of the light illuminating the opaque material. Examples ofopaque materials include molecular layers of elemental metal andpolymeric layers.

The term “binding partner” refers to a molecule that can undergo bindingwith a particular molecule. Biological binding partners are examples.For instance, Protein A is a binding partner of the biological moleculeIgG, and vice versa. Likewise, an antibody is a binding partner to itsantigen, and vice versa.

“Colloids”, as used herein, means nanoparticles, i.e. very small,self-suspendable or fluid-suspendable particles including those made ofmaterial that is, e.g., inorganic or organic, polymeric, ceramic,semiconductor, metallic (e.g. gold), non-metallic, crystalline,amorphous, or a combination. Typically, colloid particles used inaccordance with the invention are of less than 250 nm cross section inany dimension, more typically less than 100 nm cross section in anydimension, and in most cases are of about 2-30 nm cross section. Oneclass of colloids suitable for use in the invention is 10-30 nm in crosssection, and another about 2-10 nm in cross section. Colloids may beassociated with a binding partner, for example, an antibody. As usedherein this term includes the definition commonly used in the field ofbiochemistry.

As used herein, a component that is “immobilized relative to” anothercomponent either is fastened to the other component or is indirectlyfastened to the other component, e.g., by being fastened to a thirdcomponent to which the other component also is fastened, or otherwise istransitionally associated with the other component. For example, asignaling entity is immobilized with respect to a binding species if thesignaling entity is fastened to the binding species, is fastened to acolloid particle to which the binding species is fastened, is fastenedto a dendrimer or polymer to which the binding species is fastened, etc.

“Signaling entity” means an entity that is capable of indicating itsexistence in a particular sample or at a particular location. Signalingentities of the invention can be those that are identifiable by theunaided human eye, those that may be invisible in isolation but may bedetectable by the unaided human eye if in sufficient quantity (e.g.,colloid particles), entities that absorb or emit electromagneticradiation at a level or within a wavelength range such that they can bereadily detected visibly (unaided or with a microscope including anelectron microscope or the like), optically, or spectroscopically,entities that can be detected electronically or electrochemically, suchas redox-active molecules exhibiting a characteristicoxidation/reduction pattern upon exposure to appropriate activationenergy (“electronic signaling entities”), or the like. Examples includedyes, pigments, electroactive molecules such as redox-active molecules,fluorescent moieties (including, by definition, phosphorescentmoieties), up-regulating phosphors, chemiluminescent entities,electrochemiluminescent entities, or enzyme-linked signaling moietiesincluding horseradish peroxidase and alkaline phosphatase. “Precursorsof signaling entities” are entities that by themselves may not havesignaling capability but, upon chemical, electrochemical, electrical,magnetic, or physical interaction with another species, become signalingentities. An example includes a chromophore having the ability to emitradiation within a particular, detectable wavelength only upon chemicalinteraction with another molecule. Precursors of signaling entities aredistinguishable from, but are included within the definition of,“signaling entities” as used herein.

In one aspect, the invention may be used to provide a series of fluidsto a device such as a microfluidic device. The microfluidic device maybe one of those described herein or may be any other microfluidicdevice. For example, fluids may be flowed in series to a reaction sitein a microfluidic assay. The fluids may be gases, aqueous liquids, ornon-aqueous liquids. Fluids and fluid components may include, forexample, reagents, rinses, pre-rinses, fixatives and stains. The fluidsmay be flowed to one or more reaction sites with little or no mixingbetween different reagents. A series of rinse solutions may be separatedby a separation plug, allowing a first rinse solution to pass completelyover a reaction site before a second rinse solution is applied to thesite.

In one aspect, a vessel is provided to contain, store, protect and/ortransport two or more fluids. As used herein, vessels include cartridgesand tubes. A vessel may contain two or more distinct fluids separated bya third fluid that is immiscible with both. Any number of distinctfluids may be contained in a vessel. For example, FIG. 12 illustrates inlongitudinal cross-section an embodiment where the vessel is a tube 10that includes a reagent solution plug 20 followed by an air plug 30,followed by a rinse solution plug 40. An additional air plug 50 mayseparate the first rinse solution plug 40 from a second rinse solutionplug 60. The ends of the tube 70 and 72 may be sealed, for example, toretain the plugs and to prevent contamination from external sources. Theliquid plugs may retain their relative positions in the tube and may beprevented from contacting each other by the interspaced air plugs. Thetube dimensions and materials of construction may be chosen to helpfluid plugs retain their position and remain unmixed.

Reagents and other fluids may be stored for extended lengths of time inthe vessel. For example, reagents may be stored for greater than 1 day,1 week, 1 month or 1 year. By preventing contact between fluids, fluidscontaining components that would typically react or bind with each otherare prevented from doing so, while being maintained in a continuouschamber.

Fluids may be transferred from the vessel to be used in a process, forexample, to participate in a reaction or assay. Fluids may betransferred from the vessel by applying pressure or vacuum afterremoving or piercing the seal at ends 70 and 72. In other embodiments,the vessel need not be sealed and fluid flow can be started by applyingan external force, such as a pressure differential. One end of thevessel, for example, end 70, can be in or can be placed in fluidcommunication with another device that will receive the fluids from thevessel. Such a device may include, for example, a reaction site of areactor or an assay.

A vessel containing fluid plugs may be put in fluid communication with areaction site and fluids may be flowed from the vessel to the reactionsite. For instance, the fluids may be flowed to a microfluidicimmunoassay, some embodiments of which are described herein. The vesselcontaining the fluid plugs may be separate from a device including thereaction site or may be part of the same platform. Fluid may be flowedto the reaction site by, for example pushing or pulling the fluidthrough the vessel. Fluids can be pushed to the reaction site using, forexample, a pump, syringe, pressurized vessel, or any other source ofpressure. Alternatively, fluids can be pulled to the reaction site byapplication of vacuum or reduced pressure on a downstream side of thereaction site. Vacuum may be provided by any source capable of providinga lower pressure condition than exists upstream of the reaction site.Such sources may include vacuum pumps, venturis, syringes and evacuatedcontainers.

In one set of embodiments, a vessel may contain fluid plugs in linearorder so that as fluids flow from the vessel to a reaction site they aredelivered in a predetermined sequence. For example, an assay mayreceive, in series, an antibody fluid, a rinse fluid, a labeled-antibodyfluid and a rinse fluid. By maintaining an immiscible fluid (aseparation fluid) between each of these assay fluids, the assay fluidscan be delivered in sequence from a single vessel while avoiding contactbetween any of the assay fluids. Any immiscible fluid that is separatingassay fluids may be applied to the reaction site without altering theconditions of the reaction site. For instance, if antibody-antigenbinding has occurred at a reaction site, air can be applied to the sitewith minimal or no effect on any binding that has occurred.

In one embodiment, at least two fluids may be flowed in series from acommon vessel, and a component of each fluid may participate in a commonreaction. As used herein, “common reaction” means that at least onecomponent from each fluid reacts with the other after the fluids havebeen delivered from the vessel, or at least one component from eachfluid reacts with a common component and/or at a common reaction siteafter being delivered from the vessel. For example, a component of thefirst fluid may react with a chemical or biological entity that isdownstream of the vessel. A chemical or biological entity may form areaction site and may be, for example, a sample, a biological orchemical compound, a cell, a portion of a cell, a surface or asubstrate. The chemical or biological entity may be fixed in position ormay be mobile. A component from the second fluid may then react and/orassociate with the component from the first fluid that has reacted withthe downstream chemical or biological entity, or it may react orassociate with the chemical or biological entity itself. Additionalfluids may then be applied, in series, to the biological or chemicalentity to effect additional reactions or binding events or as indicatorsor signal enhancers.

Pre-filling of the vessel with reagents may allow the reagents to bedispensed in a predetermined order for a downstream process. In caseswhere a predetermined time of exposure to a reagent is desired, theamount of each fluid in the vessel may be proportional to the amount oftime the reagent is exposed to a downstream reaction site. For example,if the desired exposure time for a first reagent is twice the desiredexposure time for a second reagent, the volume of the first reagent inthe vessel may be twice the volume of the second reagent in the vessel.If a constant pressure differential is applied in flowing the reagentsfrom the vessel to the reaction site, and if the viscosity of the fluidsis the same or similar, the exposure time of each fluid at a specificpoint, such as a reaction site, may be proportional to the relativevolume of the fluid. Factors such as vessel geometry, pressure orviscosity can also be altered to change flow rates of specific fluidsfrom the vessel.

Another aspect of the invention centers around filling a vessel withfluid plugs. In one embodiment, the vessel is a tube and the tube isfilled sequentially with a series of fluid plugs separated by plugs ofimmiscible separating fluids. Fluids may be disposed in the tube in anymanner that allows two or more fluid plugs to be separated by one ormore separation fluid plugs. For example, fluids may be pumped into thetube under pressure or pulled into the tube by vacuum.

In one embodiment, a first end of the tube may be connected to a vacuumsource. The tube may be pre-filled with a fluid, such as a buffer, thatexhibits greater viscosity than air and may allow for more precisecontrol of fill rates than if the tube were simply filled with air.Some, or all, of any fluid that is used to pre-fill the tube may beexpelled from the tube during the filling process. Between the portionof the tube to be filled and the vacuum source may be placed a valvethat can be opened or closed to provide vacuum to the tube. The opposingend of the tube may be placed in a reservoir that may be, for example, avial or a well in a 96 well plate. The reservoir may contain a fluidsuch as a buffer, reagent fluid, rinse solution, precursor or separatingfluid. The valve may be opened for a time period long enough to draw inthe desired amount of fluid from the reservoir. The valve may becontrolled manually or by a controller such as computer. After the valvehas closed, the opposing end of the tube can be removed from the fluidreservoir and a second fluid plug may be drawn into the tube. If air isthe second fluid, the valve may be actuated while the end of the tube issuspended in air, not in a reservoir. When an air plug of suitablelength has been aspirated into the tube, the valve may be closed and theopposing end of the tube may be placed in a fluid reservoir that may bethe same as, or different from, the first. The valve may then be openedagain for a length of time appropriate for aspirating the desired plugsize. This may be followed by another separating fluid plug that may bethe same or different from the first. The procedure may be repeateduntil a predetermined sequence and quantity of fluids have beenaspirated into the tube. In some cases, the tube can then be sealed, atone or both ends. Multiple tubes may be aspirated in parallel using acommon controller, such as a computer. Fluids may be drawn from commonor separate vessels when more than one tube is filled.

In another embodiment, vessels such as tubes may be filled without avacuum pump or without a source of electric power. For example, ahand-operated syringe may provide a vacuum source for aspirating fluids.The syringe plunger may be withdrawn a specific distance to provide foraspiration of a specific amount of fluid into an opposing end of a tube.Valving may not be necessary. Multiple tubes may be filled in parallel.

In one aspect, a vessel may be used to retain two or more fluids thatcan be delivered in series from the vessel. The vessel may be any shapeand size and may be made of any material appropriate for retaining thefluids which it is designed to hold. Depending on the fluids, thismaterial may be, for example, glass, metal, or a polymer. Polymers mayinclude, for example, thermoplastics such as polyethylene andpolypropylene, polycarbonates, polystyrene, PTFE, PET, and others knownto those skilled in the art.

In some embodiments, the vessel is a tube. Tubes may be preferred asthey are readily available in different diameters, lengths andmaterials. Tubes may be flexible and may be translucent or transparent.Fluid plugs in a tube may be measured linearly as an indication of thevolume of the plug. The tube may have a consistent or variable innerdiameter and may have a length-to-internal diameter ratio greater than10 to 1, greater than 50 to 1, or greater than 100 to 1. Depending uponthe application, tubes of any diameter may be used, and in manyapplications the tube may have an inner diameter of less than 1 cm, lessthan 5 mm, less than 1 mm, less than 500 microns, less than 200 microns,less than 100 microns, or less than 50 microns. A tube with a greaterlength-to-internal diameter ratio may be useful in visually indicatingthe amount of each fluid contained in the tube. For instance, a linearmeasurement of a fluid plug in a tube of known inner diameter may givean accurate indication of the volume or the relative volume of thefluid.

The vessel, if a tube or another shape, may include two or more branchesor sections that may be in fluid communication with each other and withthe remaining interior of the vessel. In some embodiments, a tube mayhave two, three, four or more branches that may be interconnected. Thebranches and branch junctions may or may not include valves. Valves maybe used to temporarily segregate one or more branches, and any liquidcontained therein, from the remainder of the tube.

In one embodiment, a tube may include a “Y” shaped branch at one end,for instance, an upstream end. Each branch of the Y may contain a fluidthat reacts with the fluid in the other branch to form a third fluid.Drawing each fluid from each branch into a common tube may provide anenvironment for allowing the two fluids to react. The two branches mayjoin at a section, or lead to a section, that is of sufficientdimensions to promote turbulent flow and therefore mixing of the twofluids. For examples of different geometries, see U.S. patentapplication Ser. No. 09/954,710, which is incorporated by reference inits entirety herein.

In some embodiments, the material used for the vessel may be highlywetable. In other embodiments, however, the material used for thevessel, and in particular, the material used for the interior surface ofthe vessel, may exhibit low wetability. For example, when aqueoussolutions are to be contained in the vessel, the interior surface of thevessel may exhibit low wetability for aqueous solutions. If the interiorsurface of the vessel is less wetable, it may be less likely that afluid will flow along the surface. On a highly wetable surface, anaqueous solution may flow along the walls of the vessel and may be morelikely to come into contact with other fluids contained in the vessel. Aless wetable surface may allow for the use of higher inner diameter tolength ratios for tubes, cartridges or other vessels while maintainingdistinct fluid plugs during storage, shipment and/or use. Surface energyis indicative of the wetability of a surface and it may be preferredthat the vessel, or the interior surface of the vessel, have a surfaceenergy of less than 40 dynes/cm, less than 35 dynes/cm, less than 32dynes/cm or less than 30 dynes/cm. Some polymers that may exhibitsurface energies in these ranges include polypropylene, polyethylene andPTFE.

The vessel may also be made of a material having low adsorbancecharacteristics for fluid components that may be retained in the vessel.For example, if a vessel is to retain a fluid containing proteins, itmay be preferred to use a vessel made of a material that does not adsorbproteins. If the interior surface of the vessel does exhibit a tendencyto adsorb a component of a fluid, the surface may be pretreated toreduce that tendency. For example, a polymer surface may be treated withsurfactants such as Tween 20 or blocking proteins such as albumin and/orcasein to reduce its tendency to adsorb proteins. For other examples oftreating surfaces, see U.S. patent application Ser. No. 09/907,551,filed Jul. 17, 2001, titled “Surfaces that Resist the Adsorption ofBiological Species,” which is incorporated by reference in its entiretyherein.

The vessel may be disposable or reusable and when in the form of a tube,may be convoluted, for example in a serpentine pattern, to extend thelength that can fit in a given space.

One or more ends of the vessel may be sealed in order to protect andretain any liquids that may be stored within. Some materials, inparticular, thermoplastics and glass, may be sealed by melting the ends.Ends may also be sealed by crimping, capping, stoppering or fixing anymaterial to the end to prevent flow or evaporation of fluid from thevessel. In one embodiment, a fluid having low volatility, such as an oilor glycol may be placed in the end of a tube to help prevent evaporationand/or movement of other fluids contained therein.

In various embodiments, any type of fluid or fluids may be used. Fluidsinclude liquids such as solvents, solutions and suspensions. Fluids alsoinclude gases and mixtures of gases. When multiple fluids are containedin a vessel (such as a tube) the fluids may be separated by anotherfluid, that is preferably immiscible in each of the first two fluids.For example, if a tube contains two different aqueous solutions, aseparation plug of a third fluid may be immiscible in both of theaqueous solutions. When aqueous solutions are to be kept separate,immiscible fluids that can be used as separators may include gases suchas air or nitrogen, or hydrophobic fluids that are substantiallyimmiscible with the aqueous fluids. Fluids may also be chosen based onthe fluid's reactivity with adjacent fluids. For example, an inert gassuch as nitrogen may be used in some embodiments and may help preserveand/or stabilize any adjacent fluids. An example of an immiscible liquidfor separating aqueous solutions is perfluorodecalin. The choice of aseparator fluid may be made based on other factors as well, includingany effect that the separator fluid may have on the surface tension ofthe adjacent fluid plugs. It may be preferred to maximize the surfacetension within any fluid plug to promote retention of the fluid plug asa single continuous unit under varying environmental conditions such asvibration, shock and temperature variations. Separator fluids may alsobe inert to any reactive site to which the fluids will be supplied. Forexample, if a reactive site includes a biological binding partner, aseparator fluid such as air or nitrogen may have little or no effect onthe binding partner. The use of a gas as a separator fluid may alsoprovide room for expansion within the vessel should liquids contained inthe vessel expand or contract due to changes such as temperature(including freezing) or pressure variations.

Fluids can be transferred from a vessel for use in a chemical orbiochemical process. By applying an external force to the vessel such aspressure, suction, or g-forces, fluids may be flowed from a vessel atconstant or varying flow rates. Fluids may also be drawn from a vesselby capillary action. Pressure may be applied upstream of the fluids tobe flowed from the vessel and pressure sources may include pumps, suchas electric or manual pumps, syringes, or pressurized containers.Suction may be applied to the downstream side of the vessel by using avacuum or partial vacuum source such as a pump, syringe, evacuatedcontainer venturi or other source of reduced pressure.

In one embodiment, a vacuum source is used to flow liquids from thevessel. To control the flow of liquids from the vessel, for instance,when liquids are to be flowed over a reaction site at a specific rate,it may be preferred to apply a constant partial vacuum pressure to thedownstream side of the vessel. Accurate vacuum pressures can be providedby vacuum pump, by a portable battery-powered pump or by a syringe.Vacuum pressure less than 1.0, 0.99, 0.95, 0.9, 0.8, 0.7, 0.6, 0.5, 0.3,0.2, or 0.1 atmospheres may be used.

In some embodiments, a vacuum or partial vacuum may be applied to thevessel without the use of electrical power. For example, a syringeincluding a syringe barrel and plunger may be used to provide a sourceof vacuum. If the vessel is in communication with a reaction site, suchas that in a microfluidic assay, the vacuum source, in this case, asyringe, may be attached downstream of the reaction site. Vacuum may beapplied by placing the tip of the syringe barrel in fluid communicationwith the downstream side of the vessel retaining the fluids. If a totalvacuum, or close to total vacuum, is desired, then all air may beexpelled from the syringe by completely depressing the syringe plungerto the bottom of the barrel and subsequently attaching the barrel to thevessel. To provide less than a total vacuum, the syringe barrel may bepartially filled with air prior to withdrawing the plunger to producevacuum. For example, if a 10 cc syringe is used, a syringe barrel may befilled to 5 ccs with air and the plunger withdrawn to a total volume of10 ccs to provide a vacuum equal to one-half atmosphere. Likewise, 0.75atmosphere may be applied by filling a 10 cc syringe with 7.5 ccs of airand then withdrawing the plunger to the full 10 cc mark. A holdingdevice such as a clip or a notch in the syringe barrel may be used tohold the plunger in the constant position after it is withdrawn. If theinternal volume of the syringe used is significantly greater than thevolume of fluids to be drawn from the vessel, the vacuum pressureapplied to the vessel may be substantially constant from the beginningto the end of the drawing process. In some embodiments, the volume ofthe syringe used may be greater than 10×, 100× or 1,000× the volume ofthe fluid or fluids to be drawn.

When the syringe is filled with air to a specific volume, the air in thesyringe barrel may be at atmospheric pressure, regardless of where theprocess is performed. Compared to providing a partial vacuum at a fixedabsolute pressure, such as with an evacuated container, this manualsyringe technique may be useful under conditions of varying ambientpressure, such as at different altitudes, as the method may produce amore consistent pressure differential across the vessel and/or thereaction site, regardless of the ambient air pressure. This may aide indrawing fluids at a predetermined rate and thus subject a reaction siteto a more precise predetermined residence time for each fluid.

In another aspect, the vessel may be used to store fluids. In variousembodiments, fluids may be stored in the vessel for greater than 10seconds, one minute, one hour, one day, one week, one month, or oneyear. While they are stored, fluids may be kept separated by immiscibleseparation fluids so that fluids that would react with each other whenin contact may be stored for extended periods of time in a commonvessel. The fluids may be stored so that they are statically maintainedand do not move in relation to their position in the vessel. Fluids mayshift slightly or vibrate and expand and contract while being staticallymaintained. The common vessel may have an absence of inner walls orother dividers to keep the fluids apart and fluids may be separated bynothing more than a separation fluid. When stored in a static state, thefluids may be stored at reduced temperatures, such as less than 4° C.,less than 0° C., or less than −10° C. Fluids may also be exposed toelevated temperatures such as greater than 25° C., greater than 35° C.or greater than 50° C. Fluids may be shipped from one location to theother by surface or air without allowing for mixing of reagent fluidscontained in the vessel. The amount of immiscible separation fluid maybe chosen based on the end process with which the fluids are to be usedas well as on the conditions to which it is expected that the vesselwill be exposed. For example, if the vessel is expected to receivephysical shock or vibration, larger plugs of immiscible separation fluidmay be used. In this manner, distinct fluids within a vessel may avoidmixing.

In another embodiment, the vessel containing the fluids may be storedalong with a device including a reaction site such as an assay device ora chemical or biochemical reactor. The vessel and device may beintegrally connected or constructed and arranged to be integrallyconnected. Thus, a full reagent set may be serially lined up in thevessel and ready for application to the reaction site upon applying, forexample, pressure or vacuum to an appropriate end of the vessel ordevice.

A vessel containing a series of fluid plugs may be connected to adownstream device for participation in a chemical or biochemicalreaction. An end of the vessel, for example an end of a tube orcartridge, may be connected to a device so that the two are in fluidcommunication. This may be done directly, by inserting a tube end intoan inlet on the device or may be done indirectly by attaching via anintermediate connector, such as a length of tubing. In some cases, deadspace at the connection point may be minimized to decrease, for example,delay, mixing, or the formation of turbulent flow. The connection may bevacuum-tight. In some embodiments, the inner diameter of the connectormay be less than 1×, 3× or 5× the inner diameter of either the vessel orthe device. Some softer polymers may provide for a better connectionthan harder polymers. For example, a device made of PDMS may provide fora secure connection with minimal dead volume.

In another embodiment, a vessel including fluid plugs may be integrallyconnected to a device that includes a reaction site. For example, thevessel and device may be formed on a common platform such as amicrofluidic chip. The device and vessel may be in fluid communicationso that when a vacuum or partial vacuum is applied downstream of areaction site, fluid plugs are drawn from the vessel. Likewise, pressureapplied upstream of the fluid plugs may push the fluids into the deviceand apply them to the reaction site.

More than one vessel may be integrally connected or non-integrallyconnectable to a device including a reaction site. For example, if twovessels are connected to the upstream side of a device, the fluids inone of the vessels may be passed over the reaction site by unsealing anupstream end of the first device while maintaining a seal on theupstream side of the second device. Vacuum applied to the downstreamside of the device will draw reagents from the first vessel but not thesecond. In a similar manner, two or more devices including reactionsites may be connected to a single vessel containing fluids, and thefluids may be drawn through a chosen device by, for example, applyingvacuum to that device while leaving the other device or devices sealed.

Samples of all types may be used in conjunction with differentembodiments. Samples may include chemical samples such as water,solvents, extracts, and environmental samples. Samples may also includebiological samples such as whole blood, serum, plasma, tears, urine andsaliva. A sample being examined with an assay or reacted in a reactormay be transferred either to a reaction device or to a vessel containingreagent fluids. For example, a sample of whole blood may be placed inthe inlet of an assay device and may be flowed over the reaction site byusing vacuum or pressure. This may occur prior to connecting the vesselor prior to flowing reagents from the vessel to the reaction site.Alternatively, the sample may be placed in a vessel containing reagentfluids. For instance, a whole blood sample of measured volume may beinjected into the downstream end of a tube containing fluid reagents.The tube may then be connected to the assay device and, upon applicationof vacuum or pressure, the sample may be applied to the reaction site inadvance of the reagents that are flowed serially from the tube. Inanother embodiment, some reagents may be flowed to the reaction site,followed by a sample, which is in turn followed additional reagents. Inyet other embodiments, the sample may be flowed last.

In one embodiment, a device including a reactive site may be integrallyor non-integrally connected to a sampling device, such as a samplingtube. The sampling tube may have one end associated with a channel orchamber housing the reaction site. An opposing end of the tube may bedipped into a sample source (that may be a container or may be asubject) to be analyzed or reacted. By applying vacuum downstream of thereaction site, sample may be drawn into the sample tube and may eitherbe maintained in the sample tube or drawn to the reaction site. When apredetermined amount of sample is obtained, the end of the sample tubemay be removed from the sample source or the vacuum may be stopped. Insome embodiments, the same sample tube may then serve as a connectorbetween the device and a vessel holding reagent fluids. By placing theend of the sampling tube in fluid communication with the downstream sideof the vessel containing fluids, the fluids may be drawn through thechannel of the device in a method similar to how the sample was drawnthrough. In some cases, a first reagent fluid in the vessel may bechosen to help carry the sample, treat the sample, dilute the sample, orrinse the sample from the tube.

The invention provides a method and apparatus for determining apresence, qualitatively or quantitatively, of a component in a sample.The component may be a binding partner, such as an antibody or antigen,that may be indicative of a disease condition.

In one aspect, a sample from a subject can be analyzed with little or nosample preparation. The sample may also be obtained non-invasively, thusproviding for a safer and more patient-friendly analytical procedure.

In another aspect, an assay providing high sensitivity and a low limitof detection, comparable to that of the most sensitive ELISA test, isprovided. The assay can be run quickly and results may be permanent,allowing for reading the assay at any time after performing the test.

In another aspect, a sample is flowed over a surface associated with aprospective binding partner of a sample component. The assay can beperformed in a channel of a microfluidic device allowing the sample tobe flowed over a binding partner, for example, an antigen. Anyantigen-antibody complex that forms may be associated with a metalcolloid that provides a catalytic surface for the deposition of anopaque material, such as a layer of metal. Therefore, ifantibody-antigen binding occurs in the microfluidic channel, the flowingof a metal precursor through the channel can result in the formation ofan opaque layer, such as a silver layer, due to the presence of thecatalytic metal colloid associated with the antibody-antigen complex.Any opaque layer that is formed in the microfluidic channel can bedetected optically, for example, by measuring a reduction in lighttransmittance through the microfluidic channel compared to a portion ofthe channel that does not include the antibody or antigen. The opaquelayer may provide an increase in assay sensitivity when compared totechniques that do not form an opaque layer.

As used herein, “fastened to or adapted to be fastened”, in the contextof a species relative to another species or to a surface of an article,means that the species is chemically or biochemically linked viacovalent attachment, attachment via specific biological binding (e.g.,biotin/streptavidin), coordinative bonding such as chelate/metalbinding, or the like. For example, “fastened” in this context includesmultiple chemical linkages, multiple chemical/biological linkages, etc.,including, but not limited to, a binding species such as a peptidesynthesized on a polystyrene bead, a binding species specificallybiologically coupled to an antibody which is bound to a protein such asprotein A, which is attached to a bead, a binding species that forms apart (via genetic engineering) of a molecule such as GST or Phage, whichin turn is specifically biologically bound to a binding partnercovalently fastened to a surface (e.g., glutathione in the case of GST),etc. As another example, a moiety covalently linked to a thiol isadapted to be fastened to a gold surface since thiols bind goldcovalently. Similarly, a species carrying a metal binding tag is adaptedto be fastened to a surface that carries a molecule covalently attachedto the surface (such as thiol/gold binding) which molecule also presentsa chelate coordinating a metal. A species also is adapted to be fastenedto a surface if a surface carries a particular nucleotide sequence, andthe species includes a complementary nucleotide sequence.

A microfluidic device of the invention can be fabricated of a polymer,for example an elastomeric material such as poly(dimethylsiloxane)(PDMS) using rapid prototyping and soft lithography. For example, a highresolution laser printer may be used to generate a mask from a CAD filethat represents the channels that make up the fluidic network. The maskmay be a transparency that may be contacted with a photoresist, forexample, SU-8 photoresist (MicroChem), to produce a negative master ofthe photoresist on a silicon wafer. A positive replica of PDMS may bemade by molding the PDMS against the master, a technique known to thoseskilled in the art. To complete the fluidic network, a flat substrate,for example, a glass slide. silicon wafer, or polystyrene surface may beplaced against the PDMS surface and may be held in place by van derWaals forces, or may be fixed to the PDMS using an adhesive. To allowfor the introduction and receiving of fluids to and from the network,holes (for example 1 millimeter in diameter) may be formed in the PDMSby using an appropriately sized needle. To allow the fluidic network tocommunicate with a fluid source, tubing, for example of polyethylene,may be sealed in communication with the holes to form a fluidicconnection. To prevent leakage, the connection may be sealed with asealant or adhesive such as epoxy glue.

In one embodiment, as shown in FIG. 1, a microfluidic device 110 can beused to provide a substrate on which to perform the assay. Methods ofmanufacturing such a microfluidic device are provided in U.S. Pat. No.6,645,432, incorporated by reference in its entirety herein.

A series of microfluidic channels, 120, 122, and 124, can be used toflow sample and metal precursor across the surface 130 of themicrofluidic device. A binding partner, for example, an antigen orantibody, may be disposed on surface 130 at portion 140. Portion 140 mayinclude a stripe of binding partner, as shown, transversing two or morechannels. Alternatively, a binding partner may be disposed on a portionlimited to a single channel. Multiple binding partners may be disposedin a single channel and may overlap or be segregated from each other.

Binding partners immobilized at a region or portion of a region can beimmobilized in essentially any manner, and many immobilizationtechniques suitable for use with the invention are known in the art. SeeU.S. patent application Ser. Nos. 10/654,587 and 09/578,562, which areincorporated by reference in their entirety herein. Immobilization canbe done in a way such that the species are randomly oriented relative tothe surface (i.e., each immobilized species can be oriented, relative tothe surface, randomly), or greater control of the orientation of speciesrelative to the surface can be provided. For example, where proteins areimmobilized at the surface, they can be oriented such that their bindingsites for the assay are oriented generally away from the surface,maximizing their binding capacity or availability. One technique fordoing so, described in U.S. Pat. No. 5,620,850, incorporated herein byreference, involves synthesizing the protein with a polyamino acid tagsuch as, for example, a sequence of 6 histidines, at a locationgenerally opposite the protein's relevant binding site, providing ametal chelate, such as nitrilotriacetic acid, chelating a metal ion suchas nickel in such a way that at least two coordination sites on nickelare free for binding to the polyamino acid tag, and allowing the tag tocoordinate to the metal ions, thus immobilizing the protein at theregion or portion of a region in an advantageous orientation. A metalchelate such as this can be immobilized at the region in any of a numberof ways. One way involves forming a self-assembled monolayer (SAM) atthe region, terminating in the metal chelate, as described in theabove-referenced U.S. Pat. No. 5,620,850. For example, a thin,essentially transparent thin gold layer can be deposited at the region,and SAM-forming alkyl thiols, terminating in a metal chelate, can bedeposited on the gold layer as a SAM. Other chemistry, described in U.S.Pat. No. 5,620,850 and other references, and known to those of ordinaryskill in the art, can be used to form such a SAM on a region defined bya variety of base materials.

To run the assay, a sample, such as a biological sample taken from asubject, is flowed through one or more microchannels 120, 122, or 124,in the direction shown by the arrows. The sample may be a liquid sample,but in some embodiments need not be diluted, purified or treated priorto analysis. The sample may be flowed through the microchannel at a ratesufficient to allow a component of the sample to bind with a bindingpartner immobilized at portion 140. By actively flowing the samplethrough the channel, the reactive portion 140 is repeatedly exposed tocomponents of the sample, improving reaction kinetics and resulting inan increased formation of any binding pairs. After an adequate amount offlow of sample through microchannel 120, e.g., when detectable bindingpairs have formed, a fluid containing a metal colloid associated with asecond binding partner of the sample component is flowed to themicrochannel, allowing the metal colloid to bind with any samplecomponent that has been associated with portion 140 of the microchannel.

After the metal colloid has been given the opportunity to bind with anybinding partner at portion 140, a metal precursor can be flowed throughchannel 120 in a similar manner as was the metal colloid. The metalprecursor is flowed through the microchannel at a concentration and arate that allows an opaque layer to be formed wherever a thresholdnumber of metal colloids have been associated with the surface. Thus, ifa gold conjugated antibody is used as a metal colloid, a silver nitratesolution may be used to electrolessly deposit a silver layer on theportion of the channel associated with the gold conjugated antibody. Atthe completion of this portion of the assay, surface 130 of themicrofluidic network may include, in successive layers, an antigen suchas HIV antigen, a sample component of an HIV antibody obtained from asubject, a metal colloid such as gold-labeled anti-human IgG, and anopaque layer of metal, such as silver, that has been electrolesslydeposited on the metal colloid. Rinsing solutions may be flowed throughthe channel before or after each of the steps.

In addition to depositing metal on any metal colloids that may beassociated with portion 140 of microchannel 120, the metal precursor mayalso be deposited on metal that has previously been electrolesslydeposited on the gold-conjugated antibody. In this manner, an opaquematerial may be formed over some or all of portion 140 allowing fordetection by, for example, the unaided eye or an optical detectiondevice. The opaque material may be a continuous material rather than,for example, independent particles, and may include a horizontaldimension that, in a dimension measured in substantially the same planeas surface 130, measures greater than 1 micron, greater than 10 microns,or greater than 100 microns.

In some cases, an opaque layer may form a web or honeycomb of materialthat includes passages allowing light to be transmitted therethrough. Asadditional material is deposited, these passages may become smaller,allowing less and less light to be transmitted through the material. Asthe passages disappear, the amount of light transmittance may be reducedto zero, providing for a completely opaque material.

After an opaque layer has been formed, detection of the opaque layer,and therefore determination of the presence of a binding partner, may bedetermined by visually examining the microfluidic device or by using adetector such as an optical detector. One embodiment of an opticaldetector is depicted in FIGS. 2, 3 and 9. FIG. 2 illustratesmicrofluidic device 110, as shown in FIG. 1. Also included is lightsource 210, here an oscillator-modulated laser diode, and a detector220, such as an optical IC. As illustrated in the schematic diagram ofFIG. 3, the detector signal may be amplified and passed through abandpass filter centered at the same frequency as the oscillatorcontrolling the light source. The output may then be passed to an A/Dconverter which can then provide an output on a readout, such as an LCDdisplay. Both the light source and the detector may be powered by a 9volt battery.

In one aspect, the invention provides an apparatus and method foranalyzing a sample using continuous flow. Typically, existing methodssuch as ELISA and other sandwich assays use a 96 well plate, or similar,for containing a sample for the immunoassay. These methods can expose anantibody or an antigen to a sample component in a fluid, but the fluidis not flowed past the antibody or antigen and diffusion is relied onfor bringing binding partners into proximity with each other. Thepresent invention may allow for increased opportunities for binding of asample component to a potential binding partner at similar or lowerconcentrations of sample component than previous methods. By flowing asample containing one binding partner past a surface presenting theother binding partner, a greater number of potential binding partnersare placed in proximity to each other than would occur via simplediffusion. In one embodiment, the sample is flowed through amicro-channel providing the benefits of flowing one binding partner pasta second binding partner while requiring a small sample, for example,less than 10 micro liters, less than 1 micro liter, or less than 100nanoliters of sample. The microchannel may be of a material transparentto light that is used to detect the formation of an opaque material inthe channel so that any absorbance or transmittance of light through aportion of the channel can be attributed to the formation of an opaquelayer.

Because immunoassays detect signaling entities, such asenzyme-conjugated secondary antibodies that are dissolved or suspendedin a fluid, a relatively long path length is required in order to obtainoptimal sensitivity. Thus, one reason why immunoassays have not beenapplied in microfluidics is the short path length typically presented bymicrofluidic devices. For example, a microfluidic device may have achannel having a thickness of less than 250 microns, less than 100microns, or less than 40 microns. Therefore, any fluid filling a channelin this microfluidic device would present a perpendicular light pathwayof less than 250, 100 or 40 microns. The present method may not besubject to these restrictions because it can use an opaque layer in thesolid state, rather than a chromophore in a fluid. The opaque layer mayhave a thickness of less than 1 micron, of less than 100 nanometers orless than 10 nanometers. Even at these small thicknesses, a detectablechange in transmittance can be obtained.

The geometry of the microfluidic channel may provide for the laminarflow of fluids through the channel, even at relatively high flow rates.Alternatively, turbulent flow may be employed by using even faster flowrates or devices such as microfluidic mixers. Such mixing may providefor a greater amount of contact between potential binding partners.

The presence, absence, or amount of an analyte in a sample may beindicated by the formation of an opaque material. Although the opaquematerial may be used to refract light or may be excited to emit light ata similar or different wavelength than the light to which the layer isexposed, the measurement of light transmission may be preferred due to,for example, lower equipment and operating costs, and ease of use. Insome microchannels, an opaque layer may be visible to the naked eye and,in particular if reflective, may be detected without the use ofinstrumentation.

Any opaque material that forms can be a series of discontinuousindependent particles, but in one embodiment is a continuous materialthat takes on a generally planar shape. The opaque material may have adimension greater than 1 micron or greater than 10 microns. The opaquematerial may be a metal and is preferably a metal that can beelectrolessly deposited. These metals include, for example, copper,nickel, cobalt, palladium, and platinum. A metal precursor is a materialthat can provide the source of the elemental metal for depositing on,for example, a metal colloid. For example a metal precursor may be ametallic salt solution such as silver nitrate. In one embodiment, ametal precursor may include 0.1% silver nitrate, 1.7% hydroquinone and0.1 M citrate buffer at a pH of 3.5. Some other examples ofelectrolessly deposited materials can be found in Modern Electroplating,4^(th) Edition, Schlesinger and Paunovic, Wiley, 2000. Metal precursorscan be stored for long periods of time and may be stable for severalyears whereas optically active compounds may have much shorter shelflives.

Any metal colloid associated with a surface may be widely scattered overa portion of the surface. For example, gold conjugated antibodies may bebound to sample components that are associated with the portion of thesurface but spaces may exist between the gold conjugated antibodies,making them discontinuous. When a metal precursor is first exposed tothese gold conjugated antibodies, the precursor may form particulatescentered around individual metal colloids. As metal, e.g., silver, isdeposited on these metal colloids, the particles become larger and soonthe metal precursor may deposit metal not only on gold colloids but onmetal that has been previously electrolessly deposited. For example, asilver nitrate solution may deposit silver metal on to silver metalparticles that have previously been deposited on gold conjugatedantibodies. Thus, as the silver layer continues to grow on silver, aswell as on gold, areas that previously were independent particles orislands of metal can join to form a larger, continuous opaque materialthat can be easily detected. It has been found that a microfluidicsystem can provide for a relatively smooth, continuous layer of metal.The opaque material may have a thickness greater than 1, 10, 100 or 1000nanometers. For some opaque materials, the material may becomecompletely opaque at thicknesses greater than about 100 nm. However, insome embodiments, such as when a honeycomb or similar structure isformed, thickness in some portions may be much greater while stillallowing some light to be transmitted.

A variety of chemical and biological materials may be analyzed by themethods and apparatuses described herein. Analytes may include chemicalssuch as organic compounds and biological materials such as proteins,peptides, nucleic acids and antibodies.

Analytes include any analyte for which a binding partner can be found.Analytes that may be determined include specific proteins, viruses,hormones, drugs, nucleic acids and polysaccharides; specificallyantibodies, e.g.: IgD, IgG, IgM or IgA immunoglobulins to HTLV-I, HIV,Hepatitis A, B and non A/non B, Rubella, Measles, Human Parvovirus B19,Mumps, Malaria, Chicken Pox or Leukemia; human and animal hormones,e.g.: human growth hormone (hGM, human chorionic gonadotropin WM; drugs,e.g.: paracetamol or theophylline; marker nucleic acids, e.g.; as forgenetic finger printing analysis markers; polysaccharides such as cellsurface antigens for HLA tissue typing and bacterial cell wall material.Chemicals that may be detected include explosives such as TNT, nerveagents, and environmentally hazardous compounds such as polychlorinatedbiphenyls (PCBs), dioxins, hydrocarbons and MTBE. Typical sample fluidsinclude physiological fluids such as human or animal whole blood, bloodserum, blood plasma, semen, tears, urine, sweat, saliva, cerebro-spinalfluid, vaginal secretions; in-vitro fluids used in research orenvironmental fluids such as aqueous liquids suspected of beingcontaminated by the analyte.

In cases where an antigen is being analyzed, a corresponding antibodycan be the binding partner associated with a surface of a microfluidicchannel. If an antibody is the analyte, then an appropriate antigen maybe the binding partner associated with the surface. When a diseasecondition is being determined, it may be preferred to put the antigen onthe surface and to test for an antibody that has been produced in thesubject. Such antibodies may include, for example, antibodies to HIV.

A biological sample may be obtained noninvasively. The low level ofdetection capable with the invention allows for the use of samples thattypically contain lower concentrations of antigens or antibodies thandoes blood. For example, useful samples may be obtained from saliva,urine, sweat, or mucus. By allowing samples to be obtainednoninvasively, the methods of the invention can provide for increasedthroughput, safer sampling, and less subject apprehension.

The methods and apparatus of the present invention may be capable ofobtaining limits of detection (LOD) comparable to those achievable byimmunochromatographic assays as well as ELISA. For example,concentrations below 1 nM and even in the 100 pM range can be detected.The assay can be qualitative, quantitative, or both. As illustrated inFIG. 4, as the concentration of analyte increases, the apparentabsorbance of the opaque material increases accordingly. In FIG. 4, thesample component (analyte) is HIV antigen and the sample is human serum.Different dilutions of these sera are shown and in FIG. 6 the formationof an opaque layer indicates a positive result when compared to controlat dilutions of 1 to 10 and 1 to 100. Therefore, in addition topresence/absence type tests, a quantitative test may be provided. Such aquantitative test may be of interest, for example, to those monitoringantibody levels in a patient during treatment.

Sensitivity and Limits of Detection (LOD) of the method comparefavorably to that obtainable with various state-of-art ELISA techniques.When compared to ELISA techniques using chemiluminescence, fluorescenceand absorbance in assaying rabbit IgG, an embodiment of the inventionusing silver deposition provided comparable sensitivity and LOD numbers.Sensitivity and LOD were calculated using IUPAC definitions and areprovided in Table 1 below. Higher sensitivity numbers indicate greatersensitivity and lower LOD numbers indicate a lower LOD.

TABLE 1 Method Sensitivity (normalized) LOD (pM) Silver deposition .0889 Chemiluminescence .19 22 Fluorescence .12 163 Absorbance .04 55

In another embodiment, an assay is provided that requires less time torun than typical immuno-based assays such as ELISA. For example, using amicrofluidic device of the present invention, incubation times for eachreagent can be less than 10 minutes. For ELISA techniques usingmicrowells, 1 to 3 hours incubation time is typically required for eachreagent. Thus, the present method can provide a 6 to 18 fold decrease inincubation time. A portion of this time savings can be attributed toanalyzing a sample directly without needing to purify, dilute orotherwise prepare a sample. For example, a saliva sample may be flowedacross a channel without having been diluted, filtered, separated, orotherwise prepared. From the time a sample is obtained to when resultsare realized, a total time of less than one hour, less than 30 minutes,less than 20 minutes or less than 10 minutes may be realized. One reasonfor this increase in speed is an improved rate of binding betweenbinding partners. This can be attributed, at least in part, to the flowsystem of the invention. Systems relying on diffusion, or capillaryaction are limited in the number of binding partners that can be exposedto each other over a given time period. Furthermore, as diffusion may betemperature dependent, the present invention, utilizing sample flow, maybe more temperature independent than other methods, providing for a morerobust assay in the field where temperatures may vary from above 40° C.to below 0° C.

In another embodiment, two or more parallel assays may be run. A singlesample may be physically split into two or more samples using amicrofluidic device. A microfluidic device may have a single inputchannel that branches into two, three, or more parallel channels.Parallel analysis may be performed at different threshold levels of asimilar or identical analyte, or for different analytes at the same ordifferent thresholds. A control may also be performed in parallel. Thus,with a single sample run, a sample can be analyzed for two or moreanalytes at any number of threshold concentrations. A control may alsobe run concurrently and may be useful in calibrating and/or verifyingthe detection method that is used. Once an opaque layer is formed, theassay may be stable for an extended period of time, for example, greaterthan one month or one year, so that assays may be collected and analyzedor re-analyzed at a later date.

Reagents and samples may be supplied to the assay using methods known tothose skilled in the art, using methods described herein, or usingmethods described in United States Provisional Patent Application titled“Fluid Delivery System and Method,” filed on even date, which is herebyincorporated by reference in its entirety herein.

A variety of determination techniques may be used. Determinationtechniques may include optically-based techniques such as lighttransmission, light absorbance, light scattering, light reflection andvisual techniques. Determination techniques may also measureconductivity. For example, microelectrodes placed at opposite ends of aportion of a microfluidic channel may be used to measure the depositionof a conductive material, for example an electrolessly deposited metal.As a greater number of individual particles of metal grow and contacteach other, conductivity may increase and provide an indication of theamount of conductor material, e.g., metal, that has been deposited onthe portion. Therefore, conductivity or resistance may be used as aquantitative measure of analyte concentration.

Another analytical technique may include measuring a changingconcentration of a precursor from the time the precursor enters themicrofluidic channel until the time the precursor exits the channel. Forexample, if a silver nitrate solution is used, a silver sensitiveelectrode may be capable of measuring a loss in silver concentration dueto the deposition of silver in a channel as the precursor passes throughthe channel.

Different optical detection techniques provide a number of options fordetermining assay results. In some embodiments, the measurement oftransmission or absorbance means that light can be detected at the samewavelength at which it is emitted from a light source. Although thelight source can be a narrow band source emitting at a single wavelengthit may also may be a broad spectrum source, emitting over a range ofwavelengths, as many opaque materials can effectively block a wide rangeof wavelengths. The system may be operated with a minimum of opticaldevices. For instance, the determining device may be free of a photomultiplier, may be free of a wavelength selector such as a grating,prism or filter, or may be free of a device to direct or columnate lightsuch as a columnator. Elimination or reduction of these features canresult in a less expensive, more robust device.

In one embodiment, the light source can be pulse modulated, for example,at a frequency of 1,000 Hz. To match the pulse modulated light source, adetector may include a filter operating at the same frequency. By usinga pulse modulated light source it has been found that the system can beless sensitive to extrinsic sources of light. Therefore, the assay mayrun under various light conditions, including broad daylight, that mightmake it impractical to use existing techniques. Experimental resultsindicate that by using a pulse modulated light source and filter,results are consistent regardless of the light conditions under whichthe test is run.

The light source may be a laser diode. For example, an InGaAlP redsemiconductor laser diode emitting at 654 nm may be used. Thephotodetector may be any device capable of detecting the transmission oflight that is emitted by the light source. One type of photodetector isan optical integrated circuit (IC) including a photodiode having a peaksensitivity at 700 nm, an amplifier and a voltage regulator. If thelight source is pulse modulated, the photodetector may include a filterto remove the effect of light that is not at the selected frequency.

EXAMPLES

An experiment was designed and run to evaluate the use of aheterogeneous immunoassay in combination with a cartridge containing aseries of fluid plugs. All required reagents, except for the sample,were contained in the cartridge.

Photomasks for photolithography were obtained from PageWorks (Cambridge,Mass.). Negative photoresist SU8 was obtained from Microchem (Newton,Mass.). Poly(dimethylsiloxane) Sylgard184 (PDMS) was obtained from DowCorning (Midland, Mich.). Polystyrene substrates were purchased fromNUNC (Rochester, N.Y.). Rabbit, anti-rabbit and mouse immunoglobulin G(IgG) were purchased from Sigma (St-Louis, Mo.), and Alexa-488 donkeyanti-sheep IgG was obtained from Molecular Probes (Eugene, Oreg.). Ahand-operated vacuum pump and polyethylene tubing (Intramedic PE-60,0.76 mm internal diameter and 1.22 mm external diameter) were purchasedfrom VWR Scientific Products (Pittsburgh, Pa.). All other chemicals wereof analytical grade and are available from chemical supply houses.

Preparation of a microfluidic platform. A PDMS replica withmicrochannels was prepared by rapid prototyping as described in U.S.Pat. No. 6,645,432 and in U.S. Patent Application titled “Assay Deviceand Method,”, filed on even date herewith, both of which areincorporated by reference in their entireties herein. A microfluidicdesign was used to pattern stripes of antigen (two parallel channels,30-mm long, 200-μm wide and 60-μm deep), and a second design was used tocarry out the immunoassay (six parallel channels 50-mm long, 63-μm deep)These channels were composed of 5 sections of 10 mm each, with a widthof 500 μm next to the inlet and outlet, 50 μm in the center (where theheterogeneous immunoassay takes place) and 250 μm in the intermediarysegments. This geometry results in long channels (i.e. where the sixinlets and six outlets can be geometrically separated from each other)with a limited resistance to flow (i.e. where fluids can be pumped in ahydrodynamic flow with a minimal pressure drop). The PDMS replica forpatterning was sealed non-permanently (i.e. without plasma oxidation) tothe polystyrene substrate and the two parallel channels were filled witha solution of 50 μg rabbit IgG and a solution of 50 μg mouse IgGsolution in PBS. After a 90-minute incubation time at room temperature,the channels were emptied and rinsed twice with a fresh solution of0.05% Tween in PBS. The PDMS slab was pealed off and the polystyrenesubstrate was rinsed with deionized water (conductivity larger than 18MΩ) and dried with a nitrogen gun. Inlets and outlets were punched outin the second PDMS slab (for the immunoassay) using a sharpened medicalneedle with an outside diameter of 1.6 mm (gauge 16G1½). The holes leftin the PDMS by this modified needle were large enough to insert PE-60tubing and allowed a thigh seal between the cartridge and themicrochannel. The second PDMS slab was non-permanently sealed onto thepolystyrene substrate, with its microchannels oriented orthogonally tothe stripes of antigen. The six microchannels were filled with a freshsolution of 0.05% Tween in PBS to block the exposed surface for at leasttwo hours.

Heterogeneous Immunoassay—The cartridges were inserted into the inletsof the microfluidic channels and the outlets were connected to a sourceof vacuum. To ensure a steady source of vacuum throughout the assay, thehand-operated pump was connected to a 1 L round bottom flask, acting asballast. Six PE-60 tubes were in turn connected between the ballast andthe outlets of the microfluidic chips. The assay was initiated byoperating the pump until a −15 kPa pressure difference was achievedinside the ballast, resulting in the dispensing of the contents of thecartridges into the microfluidic channels. Fluorescence intensity on thestripe of antigen (i.e. where the immunocomplex is built) and outsidethe stripe (i.e. where non-specific binding and noise level aremeasured) were quantified and subtracted to obtain the signal intensity.

Data are presented in FIG. 22 and were compensated for daily variations,using the titration curve obtained for the 8-cm plugs as an internalstandard. Each titration curve represents the average of threeexperimental curves, obtained with an interval of one day. A sigmoidalfit for the internal standard was calculated and the data weretransformed for the sigmoidal fit with the relation y=a·x+b. The valueof the constant a and b were required to obtain a fit that is y=0 at thelowest and y=1000 at the highest concentration of anti-rabbit IgG. Allexperimental data were transformed using the relation y=a·x+b and theaverage of three data points was calculated. The data presented in FIG.22 were compensated for daily variations, using a third microfluidicchip as an internal standard. The calibrator consisted of four parallelchannels (50 mm long, 50 μm wide and 50 μm deep) filled with solution of0.5, 1, 2 and 4 μM of fluorescein in 50 mM sodium carbonate buffer pH9.55. For each daily set of measurement, a new calibrator was preparedfrom a new microfluidic chip and the appropriate solutions offluorescein. The fluorescence intensity plot vs. fluoresceinconcentration resulted in a line, which was fitted by linear regression.Each fluorescence data point obtained for the immunoassay wasindividually treated with the result of the linear fit, by subtractingthe value of the intercept and then by dividing with the slope.

Tubes, or cartridges, were prepared by cutting commercially availablepolyethylene (PE) tubing into 30-cm long units. The two methodsinvestigated for filling the cartridge are illustrated in FIGS. 13 and14. In the method depicted in FIG. 14 up to six PE cartridges wereconnected to an array of voltage-gated valves, which were in-turnconnected to a −6 kPa vacuum source. Voltage pulses generated via ahome-written Labview program operated the voltage-gated valves. Beforeinitiating the reagent loading, the cartridges were filled with washingbuffer (0.05% Tween® 20 in PBS) and the valves were activated to pump a2-cm plug of air into the cartridges. The cartridges were then dipped inthe appropriate liquid and a plug of liquid was aspirated into thecartridge by opening the valves. An opening time of 5 sec resulted in a3-cm long plug or a volume of about 13.5 μL. Between each liquid plug,air was aspirated into the cartridge to separate physically each reagentand to avoid mixing of reagents in the cartridge. The valves andcartridges were affixed to a holder, which fits onto a 96-well plate,and the stock solutions of reagent were placed in the appropriatesequence into the wells. Using this system, plugs of reagents werefilled accurately in six parallel cartridges with a precision of below±14% in terms of plug length.

The second method, depicted in FIG. 14, used a manually operatedHamilton syringe connected at one end of a single length of PE tubing.This approach may be technically simpler than the method above, and maybe more appropriate for the preparation of a limited number ofcartridges. When the cartridge preparation was completed, the tubing endwas heat-sealed, the cartridge was unplugged from the valve/syringe andthe other end was heat-sealed. Cartridges containing as many as 10 plugsfor a heterogeneous immunoassay were prepared.

The air plugs located between each fluid plug ensured that no mixingbetween different reagents occurred in the cartridge. Using dyed plugs,it was observed that the plugs left small residues as they moved intothe tubing. The trailing plugs were found to collect the residue. Thisprocess resulted in a plug-to-plug cross-contamination. Multiple,distinct plugs may be more effective at removing this residue. Toquantify the cross-contamination and to determine how many rinsing plugsmight reduce the residue to acceptable levels, a cartridge was filledwith plugs of fluorescein dissolved in 50 mM carbonate buffer (pH 9.5)using the method depicted in FIG. 14. Plugs of fluorescein were loadedat various concentrations followed by one or more plugs of buffer. Thecartridge was connected to the inlet of a 25-mm long, 50×50-μmmicrofluidic channel and the fluorescence intensity in the channel wasrecorded as the plugs were pumped through the microfluidic channel.Results are shown in FIGS. 16 b and 16 c. The detector showed a linearresponse for the dilution series of fluorescein, indicating that thedata could be used for quantitative treatment. Quantification of theextent of plug-to-plug contamination trailing from a 31 μM fluoresceinsolution in three following buffer plugs showed (after backgroundsubtraction) a concentration by fluorescence of 7%, 0.9% and 0.1%relative to the 31 μM fluorescein plug. The cross-contamination from aplug of 250 μM fluorescein was measured. The presence of fluorescein inthe six plugs following the 250 μM fluorescein solution was determined.While the signal of the 250 μM fluorescein plug saturated the detector,fluorescence was detected only in the first three plugs (to follow thefluorescein plug). These observations showed that three buffer plugsbetween each reagent plug were sufficient to preventcross-contamination. Therefore, the introduction of three or more bufferplugs in the cartridge may serve a dual function: (i) preventingplug-to-plug contamination in the cartridge, and (ii) rinsing thesubstrate before the washing step, as may be desirable in applicationssuch as micro-titer assays.

In addition to air, perfluorodecalin (PFD) was evaluated as a separatingfluid. The aqueous solutions wet the hydrophilic microchannels more thandid PFD. When a PFD plug entered the microfluidic channel, it onlypartially flushed the aqueous solution out of the microchannel. Theresulting incubation times and washing efficiency were thusirreproducible. Gaseous plugs travel through the microchannel morequickly than PFD plugs do at a given pressure differential. At ambienttemperature, air has a viscosity of ˜20 μPa·s, whereas the viscosity ofPFD is ˜5 mPa·s, or 250 times larger than that of air. Upon applicationof a pressure gradient, the volumetric flow rate through channel isinversely proportional to the viscosity of the fluid. A plug of air willtherefore travel about 250 times faster than a plug of PFD. Asillustrated in FIG. 15, air plugs in the cartridge have a similar lengthas do the reagent plugs. However, due to the fast transport of air, thedetector read-out indicates that the time elapsed between twofluorescein plugs is less than 10 seconds. For comparison, a separationplug of similar length made of a liquid immiscible with water, such asPFD, took several minutes to travel through the microchannel. The totalassay time may therefore be significantly reduced when gas-basedseparators are used instead of liquid-based separators between thereagent plugs.

To illustrate the use of a cartridge for the sequential delivery ofreagents, a microfluidic heterogeneous immunoassay for the detection ofimmunoglobulin G (IgG) raised against rabbit IgG (anti-rabbit) wasperformed. A cartridge was prepared as described above with a sequenceof plugs containing sheep anti-rabbit IgG, 0.05% Tween in PBS,Alexa488-labeled donkey anti-sheep IgG and 0.05% Tween in PBS, with eachantibody-containing plug followed by three plugs of 0.05% Tween in PBS(see FIG. 17). The heterogeneous immunoassay used was similar to othersdescribed herein and in co-pending U.S. Patent Application titled “AssayDevice and Method,”, filed on even date herewith and incorporated byreference in its entirety herein, filed one even date herewith. A PDMSreplica was sealed onto an unstructured polystyrene substrate. Twoparallel microchannels were filled, one with a solution of rabbit IgGand the other with a solution of mouse IgG. After incubation, the PDMSslab was removed to reveal the bands of rabbit IgG and mouse IgGphysisorbed on polystyrene substrate. To perform the immunoassay, asecond PDMS slab with six microfluidic channels was placed orthogonal tostripes of antigen (see FIG. 18) and the surface of the polystyrene inthe microchannel was blocked with 0.05% Tween 20 in PBS. The cartridgewas connected to the microchannel inlet and a −15 kPa vacuum was appliedat the outlet using a hand-operated vacuum pump. The reagents weresequentially delivered to the microfluidic platform. Observation in themicrochannel by fluorescence microscopy revealed a clear fluorescencesignal originating from the fluorescent immunocomplex built on therabbit IgG stripe (see FIG. 20), and no fluorescence was detectedoutside the mouse IgG stripe. Some non-specific binding was observed onthe surface of mouse IgG when samples with high concentration of proteinwere used. The total assay time to perform the immunoassay could beadjusted to between 2 and 8 minutes, depending on the length of theplugs in the cartridge (see below).

The dynamic range of the heterogeneous immunoassay of anti-rabbit IgGwas determined by performing the assay with a series of 10-folddilutions of anti-rabbit IgG in the cartridges. The incubation time ofthe sample has a direct influence on of the performance of the assay.The dynamic range achieved using the technique described above wasdetermined by recording the signal as a function of the incubation time.Since the length of the plug may be directly proportional to theincubation time, cartridges were prepared, where the length of theantibody-containing plugs was varied from 2 to 4 and 8 cm. In thecartridges, both antibody-containing plugs were followed by threerinsing plugs (0.5 cm, 2.3 μL) and one washing plug (1 cm, 4.5 μL)containing 0.05% Tween in PBS (see FIG. 21). The specific incubationtimes for the antibody-containing plug and the total assay time aregiven in Table 2, below. The maximum signal increased when longerincubation times were used, and the dynamic range when using 8-cm longplugs was displaced by about one order of magnitude toward the lowsample concentrations compared to the assay with 2-cm long plugs.

TABLE 2 Plug length in cartridges and corresponding assay time Pluglength^(§) Plug volume^(§) Plug delivery time^(§) Total assay time 2 cm 9 μL 30 s 2′ 00″ 4 cm 18 μL 67 s 3′ 50″ 8 cm 36 μL 130 s  7′ 45″

A cartridge may serve as long-term storage for reagents before an assayis performed. The long-term stability of the reagents in the cartridgeswas evaluated, by preparing about 70 cartridges loaded with a sample of167 nM anti-rabbit IgG and using them over the course of 12 days. Theantibody-containing plugs were 8 cm-long. Immediately after thecartridge preparation, the assay was performed on two parallel chipswith 6 cartridges and the fluorescence signal arising from theimmunocomplex was recorded. The remaining cartridges were split intothree batches, each stored at a different temperature: 4° C., roomtemperature, and 37° C. Later, the immunoassay was repeated on twofreshly prepared chips with four cartridges from each batch. The systemillustrated in FIG. 18 can accommodate up to six cartridges in parallel.Two cartridges from all batches were used on each chip, and the averagefluorescence signal as a function of the time of storage was plotted(see FIG. 23). A slight decrease in signal was observed (see the solidline in FIG. 23, representing the average fluorescence intensityrecorded for all 12-data points). On a given chip, small variations(i.e. typically less than 5%) were observed between the signals obtainedfrom the cartridges stored at different temperatures. However, thechip-to-chip variations were larger (sometimes as high as 25%) than thecartridge-to-cartridge variations on the same chip. This suggests thatthe procedure for the preparation of the microfluidic platform may bethe source of the reduced reproducibility of the immunoassay results. Acomparison of the signal obtained with cartridges stored at differenttemperatures but used on the same chip can, however, reliably assess theloss of signal associated with the storage conditions. From the graphicsillustrated in FIG. 23, it was found that the cartridges can be storedat temperatures as high as 37° C. for two weeks without significant lossof activity compared to those stored at 4° C. or 20° C. After fourweeks, the cartridges stored at 37° C. showed a decrease in activitycompared to those stored at 4° C. or 20° C. The overall activity of theimmunoreagents in cartridges stored at 20° C. remained essentiallyunchanged for extended period of times. This was determined by comparingthe titration curves obtained for anti-rabbit IgG using freshly preparedcartridges and cartridges stored for 4 weeks at 20° C., 37° C. and 4° C.

To compare a method of the invention with existing methods, anexperiment was designed to assay HIV antibodies using the present methodas well as ELISA techniques employing chemiluminescence, fluorescenceand absorbance. Procedures and results are described below.

Reagents and equipment were obtained as follows. Rabbit IgG, anti-rabbitIgG (horseradish peroxidase conjugated), anti-rabbit IgG (alkalinephosphatase conjugated), anti-rabbit IgG (gold conjugated),p-nitrophenylphosphate (pNPP), and the silver enhancement kit wereobtained from Sigma-Aldrich (St. Louis, Mo.). AttoPhos was purchasedfrom Promega Corp. (Milwaukee Wis.). SuperSignal ELISA Femto Max waspurchased from Pierce (Rockford, Ill.). BluePhos phosphatase substratewas purchased from KPL (Gaithersburg, Md.). HIV Env antigen (gp41) waspurchased from Research Diagnostics (Flanders, N.J.). HIV positive serumand control serum were purchased from Golden West Biologicals Inc.(Temecula, Calif.).

Immunoassays in 96-well microtiter plates were performed using a TecanGenesis liquid handling robot (Center for Genomics Research, HarvardUniversity). The following Nunc MaxiSorp polystyrene plates were usedfor the silver reduction and ELISA assays: clear plates for silverreduction and absorbance, black plates for fluorescence and white platesfor chemiluminescence. Rabbit IgG (70 μL for each well) in ten-folddilutions (10 μg/mL to 100 μg/mL, which corresponded to 67 nM to 670 fM)was added to the microwells, except for one row to which PBS was addedas a negative control; incubation time was 2 hours. Blocking buffer (100μL of 0.05% Tween-20 and 1% BSA in PBS) was then added, and left toincubate for 30 minutes. For secondary antibodies, dilutions (30 μL of0.05% Tween-20 in PBS) of 1:300 anti-rabbit IgG (gold-conjugated),1:1000 anti-rabbit IgG (alkaline phosphatase), and 1:1000 anti-rabbitIgG (horseradish peroxidase) were used; incubation time was 1 hour. ForELISA substrates, pNPP (100 uL; 3 minute incubation), AttoPhos (100 uL,used within 1 week of opening; 10 minute incubation), and SuperSignalFemto ELISA (100 uL; after 5 minutes). For silver enhancement, thesolutions of silver and initiator (at 4° C.) were mixed in a 1:1 ratioimmediately before development; it was filtered through a 0.2 μm filter,and 100 uL was added to each well. After a 20 minute incubation, thesilver enhancer solution was removed, and each well was washed withwater. In general, warming the silver enhancement solution from 4° C. toroom temperature increased the rate of silver deposition. In between theaddition of each new reagent, each well was washed three times with PBS,with the following exception: deionized water was used to wash the wellsafter incubation with anti-rabbit IgG (gold) and before silverenhancement, in order to avoid precipitation of AgCl. The plate readersused were Spectramax Plus 384 for absorbance measurements, andSpectramax Gemini XS for fluorescence and chemiluminescencemeasurements.

The output of the optical IC was light transmittance; apparentabsorbance values were calculated using the relation A=−log(T/T₀), whereA is the absorbance, and T and T₀ are the transmission of the lightthrough the sample and reference, respectively, to the photodetector.Air was used as the reference in the plate reader, and a blankpolystyrene plate was used as the reference for the portable detector.

The absorbance, fluorescence, and chemiluminescence readings (y) werefit to sigmoidal curves using the software Kaleidagraph and thefollowing equation: y=Ax^(n)/(B+x^(n))+C, where x is the concentrationof the analyte, and A, B, C and n are floating parameters. Results areillustrated in FIG. 7. This equation describes a general sigmoidal curvewith the lowest possible number of floating parameters (four). Curvefitting to all four titrations gave correlation coefficients of over0.99. The readings y for all four titrations were normalized to the samescale (0 to 1) by linearly transforming each data set to achieve thevalues of A=1 (asymptote as x approaches infinity) and C=0(y-intercept).

Limits of detection were calculated according to the IUPAC definition:three times the standard deviation of the blank sample (“noise”) dividedby the slope (“sensitivity”). In samples with no rabbit IgG (i.e.negative controls), the methods that exhibited the least to most noisewere (after normalization of the signal from 0 to 1): 0.006 forabsorbance of pNPP, 0.014 for chemluminescence of SuperSignal ELISAFemto Max, 0.023 for silver (using the portable detector), and 0.066 forfluorescence of AttoPhos. The methods that showed the highest to lowestsensitivities, which were measured as slopes of the best-fit curves inthe middle of the linear working range of detection (signal of 0.50),were (in normalized units per 100 pM of analyte): 0.193 forchemiluminescence, 0.121 for fluorescence, 0.078 for silver, and 0.035for absorbance.

To prepare immunoassay samples for analysis by AFM, holes (4 mm indiameter) were punched in a PDMS slab, and the PDMS slab was placed ontoa polystyrene surface. Immunoassays were carried out in individual PDMSwells. After silver development, the PDMS slab was removed, and thesamples on the flat polystyrene substrate were analyzed by tapping modeAFM. AFM was performed with a Dimension 3100 Scanning Probe Microscope(Digital Instruments, Santa Barbara, Calif.) in tapping mode, usingsilicon probes (Si #MPP-111000; NanoDevices, Santa Barbara, Calif.) at ascan rate of 0.35 Hz. AFM micrographs are provided in FIG. 8. Streakingwas observed for samples with the largest silver grains, which suggestedthat the silver grains were loosely bound to the surface.

The microfluidic device was fabricated in PDMS using publishedprocedures in soft lithography. The dimensions of the microchannels were2 mm in width and 130 μm in height. The polystyrene surface wasinitially patterned with a stripe of HIV Env antigen (10 μg/mL) byfilling a PDMS channel (conformally sealed onto the polystyrene plate)with the antigen solution. After an overnight incubation, the channelwas emptied, the PDMS slab removed from the polystyrene surface, andrinsed the surface with deionized water. The stripe of antigen wascovered with an unstructured slab of PDMS, and oxidized the remainingsurface of polystyrene with oxygen plasma. After removal of theplasma-protective PDMS slab, another microfluidic channel (also freshlyplasma-oxidized) was sealed orthogonally to the antigen stripe. Thedimensions of these microchannels were 2 mm in width and 40 μm inheight; the width of the channel must be large enough to register asignal with the portable detector. To avoid sagging of the PDMS, pillars(which took up 12% of the surface area) were included in the channeldesign. The anti-HIV antibody assay was carried out in the microfluidicchannels with the following incubation times: 1 to 4 hours for blocking,10 minutes for samples, 10 minutes for gold-labelled anti-human IgG, and13.5 minutes for silver enhancement solution. After 6.5 minutes ofsilver enhancement, the silver solution was exchanged with a freshlyprepared one. The PDMS microchannel was removed above the initial stripeof antigen before measuring the optical density of the silver film. TheHIV assay in microwells were performed with the following incubationtimes: overnight for HIV Env antigen, 2 hours for blocking, 3 hours forsamples, 1 hour for gold-labelled anti-human IgG, and 10 minutes forsilver enhancement solution.

For each concentration of rabbit IgG and each dilution of human serum,triplicates of the immunoassay were performed, and average values andstandard deviations were calculated.

The electronic circuit consisted of a transmitter section and a receiversection. In the transmitter section, a 1 kHz oscillator modulated thelight output of a laser diode. A red semiconductor laser diode (SharpGH06510B2A; normally used for optical data storage applications such asDVD) was used; it emitted at a wavelength of 654 nm with a maximum powerof 10 mW. The laser output went through the sample to the receiversection. An optical IC (Sharp IS455; normally used in photocopymachines) to detect and amplify the signal. IS455 provided a linearoutput current with respect to the input illuminance (1 μA per lux).(The dimensions and costs of the red laser diode and the optical IC were5.6 mm and $10, and 5.0 mm and $2, respectively.) The signal was thenfiltered by a second-order bandpass filter centered at 1 kHz, and itsamplitude registered by a peak detector. The output of the peak detectorwas connected to an Analog/Digital converter that also encoded theoutput into binary coded decimal (Intersil ICL7106). The signal wasdisplayed by a 3.5 digit liquid crystal display, which provided anoutput readout range from 0 to 1999. The entire circuitry was operatedwith either a 9 V battery or a single polarity 5 V source, which wasinverted with a CMOS voltage converter (Intersil ICL7660) to create a ±5V supply. To reduce the noise in the system, pulse modulation of theoptical signal at 1 kHz was used to filter the noise power in thefrequency spectrum; as a result, only the portion of the optical noisethat fit in the pass band of the receiver filter contributed to theoverall noise detected. The system could also be used without the signalmodulation (i.e. at direct current)

The laser diode and optical IC were placed on two separate circuitboards that were held at a fixed orientation to ensure consistentalignment of the light path from the light source to the photodetector.Between the light source and photodetector, a glass plate was placed. Ablack transparency, with a pinhole aligned with the light path, wasplaced on the glass plate to block the transmission of stray light thatdid not enter the sample. To record a measurement, a polystyrene plate(either a 96-well plate or a plate with a microfluidic device) wasplaced onto the glass plate. The sample was aligned to the light path byroughly placing the sample over the pinhole, and finely adjusting the xand y position of the polystyrene plate until a maximum transmittancewas achieved. The reading from the liquid crystal display was recorded.

To compare two detection methods independent of analyte, microwells of a96 well plate were subjected to readings by an IC and by a commercialplate reader.

Absorbance of microwells containing different concentrations ofBluePhos, which absorbs maximally at 600 nm, as measured by a UV-visibleabsorbance plate reader and the optical IC described in this study. Adirect ELISA was performed on 0.67 pM to 0.67 nM of rabbit IgG as theanalyte, using an anti-rabbit IgG conjugated to alkaline phosphatase andBluePhos as the phosphatase substrate. Results are provided in FIG. 10and FIG. 11. Measurements with both devices were made at 654 nm. Thebest fit line by linear regression is shown (correlation coefficient of0.998, slope of 1.01, y-intercept of 0.08). Error bars are standarddeviations of measurements of three different microwells.

In this assay, in which the colorimetric product is a homogeneoussolution in the microwell, the two detection methods resulted in almostperfect agreement (correlation coefficient of 0.998). Thus,inhomogeneity of silver deposition on the surface may have contributedto the imperfect agreement between the two measurement methods, suchthat different parts of the same well were sampled by the laser diodeand by the plate reader (correlation coefficient of 0.996).

Example 2

A schematic representation of one embodiment and an optical detectiondevice is provided in FIG. 9. (A) Red light from the laser diode passesthrough the silver-coated microwell containing the sample to the opticalIC. A pinhole was used to block stray light that did not pass throughthe sample. The laser diode and the optical IC were driven by the samecircuit, which also had an integrated liquid crystal display that showedthe measured transmittance value.

Example 3

FIG. 10 provides a comparison of readings of an immunoassay using anoptical IC and a UV-visible plate reader. An immunoassay using silverreduction was performed on a 96-well plate that detected rabbit IgG.Optical micrographs of the silver films on microwells are shown for eachsample. The apparent absorbance of each microwell was measured by anoptical IC, and compared to its reading by a UV-visible plate reader;both measurements were made at 654 nm. The best-fit line by linearregression has a correlation coefficient of 0.989, slope of 1.12, andy-intercept of 0.16.

Having thus described several aspects of at least one embodiment of thisinvention, it is to be appreciated various alterations, modifications,and improvements will readily occur to those skilled in the art. Suchalterations, modifications, and improvements are intended to be part ofthis disclosure, and are intended to be within the spirit and scope ofthe invention. Accordingly, the foregoing description and drawings areby way of example only.

1. A method comprising: providing a first and a second fluid maintainedseparately from each other by a third fluid in a common, sealed vessel,wherein the third fluid is adjacent to and substantially immiscible withthe first and second fluids; unsealing the vessel; transferring thefirst, third and second fluids in series from the vessel to a reactionsite; and avoiding substantial contact between the first and secondfluids, at least until after the fluids have been applied to thereaction site.
 2. A method comprising: unsealing a sealed vesselcontaining a first and a second fluid maintained separately from eachother by a third fluid adjacent to and substantially immiscible with thefirst and second fluids; applying in series the first, third, and secondfluids to a reaction site, wherein the vessel and the reaction site areformed in a microfluidic chip; and avoiding substantial contact betweenthe first and second fluids, at least until after the fluids have beenapplied to the reaction site.
 3. A method comprising: unsealing a sealedvessel containing a first and a second fluid maintained separately fromeach other by a third fluid adjacent to and substantially immisciblewith the first and second fluids; applying in series the first, third,and second fluids to a reaction site, wherein the vessel and thereaction site are integrally connected to one another; and avoidingsubstantial contact between the first and second fluids, at least untilafter the fluids have been applied to the reaction site.
 4. The methodof claim 1 further comprising connecting the vessel to a devicecomprising the reaction site.
 5. The method of claim 1 wherein thevessel and reaction site are on a common platform.
 6. The method ofclaim 1 wherein the vessel and reaction site are integrally connectedduring storage of the first, second and third fluids in the vessel. 7.The method of claim 1 wherein the vessel comprises a tube.
 8. The methodof claim 1 further comprising applying a pressure differential acrossthe reaction site.
 9. The method of claim 8 wherein the pressuredifferential is provided by suction on a downstream side of the reactionsite.
 10. The method of claim 8 wherein the pressure differential isprovided by a pump on an upstream side of the reaction site.
 11. Themethod of claim 1 wherein the first and second fluids are transferred inseries to the reaction site without actuating a valve.
 12. The method ofclaim 1 wherein the first and second fluids are transferred in series tothe reaction site without actuation of any device that controls therate, the order, or timing of introduction of either of the first andsecond fluids, relative to each other, to the reaction site.
 13. Themethod of claim 4 wherein the device is a microfluidic device.
 14. Themethod of claim 1 wherein at least one of an antibody or an antigen isassociated with the reaction site.
 15. The method of claim 1 wherein thethird fluid is a gas or a gaseous mixture.
 16. The method of claim 1wherein the first fluid and/or second fluid is a rinse solution.
 17. Themethod of claim 4 further comprising disposing a sample in the deviceprior to applying the first and second fluids to the reaction site. 18.The method of claim 1 wherein the vessel contains a fourth fluid, themethod further comprising combining the fourth fluid and the secondfluid while transferring the first, third, and second fluids from thevessel to the reaction site.
 19. The method of claim 1 wherein thevessel has a length to inner diameter ratio of at least 10:1.
 20. Themethod of claim 1 wherein the vessel has an inner diameter of less than1 millimeter.
 21. The method of claim 1 wherein the vessel has an innerdiameter of less than 500 microns.
 22. The method of claim 1 wherein oneof the fluids comprises a gold conjugated antibody.
 23. The method ofclaim 1 wherein one of the fluids comprises a metal precursor.
 24. Themethod of claim 23 further comprising electrolessly depositing metal atthe reaction site to produce an opaque material.
 25. The method of claim24 further comprising determining light absorbance or transmissionthrough the opaque material.
 26. The method of claim 1, wherein thefirst and second fluids are maintained separately from each other by thethird fluid in the common, sealed vessel for greater than one hour. 27.The method of claim 2 wherein at least one of an antibody or an antigenis associated with the reaction site.
 28. The method of claim 2 whereinthe third fluid is a gas or a gaseous mixture.
 29. The method of claim 2wherein the first fluid and/or second fluid is a rinse solution.
 30. Themethod of claim 2 further comprising disposing a sample in themicrofluidic chip prior to applying the first and second fluids to thereaction site.
 31. The method of claim 2 wherein the vessel has a lengthto inner diameter ratio of at least 10:1.
 32. The method of claim 2wherein the vessel has an inner diameter of less than 500 microns. 33.The method of claim 2 further comprising electrolessly depositing metalat the reaction site to produce an opaque material.
 34. The method ofclaim 33 further comprising determining light absorbance or transmissionthrough the opaque material.
 35. The method of claim 2 wherein thevessel contains a fourth fluid, the method further comprising combiningthe fourth fluid and the second fluid while transferring the first,third, and second fluids from the vessel to the reaction site.
 36. Themethod of claim 33 wherein at least one of the first, third, and secondfluids comprises a metal precursor.
 37. The method of claim 2 whereinthe reaction site is adapted for allowing a binding event to occurbetween at least two binding partners, and wherein at least one of thebinding partners comprises an antibody.
 38. The method of claim 1wherein the reaction site is adapted for allowing a binding event tooccur between at least two binding partners, and wherein at least one ofthe binding partners comprises an antibody.
 39. The method of claim 3wherein the third fluid is a gas or a gaseous mixture.
 40. The method ofclaim 39 wherein the first fluid and/or second fluid is a rinsesolution.
 41. The method of claim 3 wherein the vessel has a length toinner diameter ratio of at least 10:1.
 42. The method of claim 41wherein the vessel has an inner diameter of less than 500 microns. 43.The method of claim 3 wherein at least one of the first, third, andsecond fluids comprises a metal precursor.
 44. The method of claim 43further comprising electrolessly depositing metal at the reaction siteto produce an opaque material.
 45. The method of claim 44 furthercomprising determining light absorbance or transmission through theopaque material.
 46. The method of claim 39 wherein the vessel containsa fourth fluid, the method further comprising combining the fourth fluidand the second fluid while transferring the first, third, and secondfluids from the vessel to the reaction site.
 47. The method of claim 3wherein the reaction site is adapted for allowing a binding event tooccur between at least two binding partners, and wherein at least one ofthe binding partners comprises an antibody.
 48. The method of claim 18wherein the fourth fluid is stored in the vessel prior to the unsealingstep.